Dengue virus infection is an emerging disease and an expanding global health problem. More than one-third of the world's population is at risk for transmission in tropical and subtropical areas and vaccines are an urgent public health priority. Although dengue rarely occurs in the continental United States, it is endemic in Puerto Rico, and in many popular tourist destinations in Latin America and Southeast Asia. Vaccines are therefore also required for individuals traveling to areas where dengue is endemic. A major problem in developing vaccines for dengue is the existence of four co-circulating serotypes. Sequential infections by different serotypes can cause an enhanced disease known as dengue hemorrhagic fever. Ideally, dengue vaccines should protect against all serotypes simultaneously or within a short time period. The most advanced dengue vaccine in clinical testing is a complex mixture of four live-attenuated viruses that requires an immunization schedule covering 12 months to achieve full seroconversion. The envelope (E) protein of dengue virus is the main determinant of virulence and is the major target of neutra- lizing and enhancing antibodies. The E protein, along with the smaller membrane (M) protein can form virus- like particles (VLPs). Altravax possesses several novel dengue envelope variants that, as DNA vaccines, can individually induce neutralizing antibodies to all four dengue serotypes in monkeys. Beginning with these novel tetravalent dengue E protein sequences, we propose to produce recombinant VLPs as vaccine candidates. VLPs are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. Past experience with approved vaccines (HBV, HPV) has shown that recombinant VLPs are potent immunogens and offer a level of safety that is difficult to achieve with live-attenuated viruses. We will evaluate mammalian, insect, and yeast systems for optimal expression of dengue VLPs. Care will be taken at this early stage to ensure that the cell lines, systems, and methods developed can ultimately be transferred to GMP manufacture. We will prepare monoclonal antibodies both to study the mechanism of tetravalent immunogenicity and to provide reagents for manufacturing controls for later work. We will evaluate VLP preparations in mice with several different adjuvant systems. Such additives can influence the isotypes of antibodies produced as well as stimulate more potent neutralizing antibody responses. Since a critical part of dengue vaccine development concerns possible disease enhancement, which is thought to be antibody- dependent in humans, we will undertake a collaboration destined to investigate these phenomena in a mouse line (AG129) that is an in vivo model of antibody-dependent severe dengue disease. The ultimate objective is to develop a safe preventative vaccine for dengue based on a single recombinant tetravalent VLP-based immunogen in a highly immunogenic format. To assist us in this work, we have assembled a team of consul- tants and collaborators with expertise in many different fields of dengue virology, immunology, and pathology. PUBLIC HEALTH RELEVANCE: Dengue virus infection is an expanding global health problem and as many as 100 million people are infected yearly. Current vaccine candidates are complex mixtures that include all four dengue types. We propose to develop a much simpler single-component vaccine using novel tetravalent immunogens.